Upload
others
View
15
Download
0
Embed Size (px)
Citation preview
© 2012 Basu et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.
International Journal of Nanomedicine 2012:7 6049–6061
International Journal of Nanomedicine
Colloidal gold-loaded, biodegradable, polymer-based stavudine nanoparticle uptake by macrophages: an in vitro study
Sumit Basu1,2
Biswajit Mukherjee1
Samrat Roy Chowdhury1
Paramita Paul1
Rupak Choudhury3
Ajeet Kumar1
Laboni Mondal1
Chowdhury Mobaswar Hossain1
Ruma Maji1
1Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India; 2Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, USA; 3Department of Biochemistry, Ballygunge Science College, Kolkata, India
Correspondence: Biswajit Mukherjee Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India Tel/fax +91 33 2414 6677 Email [email protected]
Objective: We describe the development, evaluation, and comparison of colloidal gold-loaded, poly(d,l-lactic-co-glycolic acid)-based nanoparticles containing anti-acquired immunodeficiency
syndrome drug stavudine and uptake of these nanoparticles by macrophages in vitro.
Methods: We used the following methods in this study: drug-excipient interaction by Fourier transform infrared spectroscopy, morphology of nanoparticles by field-emission scanning elec-
tron microscopy, particle size by a particle size analyzer, and zeta potential and polydispersity
index by a zetasizer. Drug loading and in vitro release were evaluated for formulations. The best
formulation was incorporated with fluorescein isothiocyanate. Macrophage uptake of fluorescein
isothiocyanate nanoparticles was studied in vitro.
Results: Variations in process parameters, such as speed of homogenization and amount of excipients, affected drug loading and the polydispersity index. We found that the drug was
released for a prolonged period (over 63 days) from the nanoparticles, and observed cellular
uptake of stavudine nanoparticles by macrophages.
Conclusion: Experimental nanoparticles represent an interesting carrier system for the trans-port of stavudine to macrophages, providing reduced required drug dose and improved drug
delivery to macrophages over an extended period. The presence of colloidal gold in the particles
decreased the drug content and resulted in comparatively faster drug release.
Keywords: stavudine, poly(d,l-lactic-co-glycolic acid), nanoparticles, colloidal gold, uptake by macrophages
IntroductionOptimization of the pharmacological action of a drug along with reduction in its toxic
side effects is a prime prerequisite for an ideal drug-delivery system. Colloidal drug car-
riers can provide site-specific or targeted drug delivery along with optimal drug release.1
Among these carriers, nanoparticles and liposomes have been widely investigated. Due
to various technical problems, such as poor stability and low entrapment efficiency of
liposomes, polymeric nanoparticles were proposed as a suitable alternative. One of
the most attractive areas of research using polymeric nanoparticles is the controlled
delivery of drug following parenteral, oral, pulmonary, nasal, and topical routes of
administration. Polymeric nanoparticles can also be targeted to specific cells and tis-
sues in the body by virtue of their small size and by functionalizing their surface with
polymers and appropriate ligands.2 Further, polymeric nanoparticles usually overcome
stability issues of liposomes and can minimize the therapeutic dose and thus minimize
drug-induced side effects by sustained drug release.3 A diverse range of materials has
been used as drug carriers, including polymers4 and dendrimers,5 and nanomaterials
Dovepress
submit your manuscript | www.dovepress.com
Dovepress 6049
O R I g I N A L R E S E A R C H
open access to scientific and medical research
Open Access Full Text Article
http://dx.doi.org/10.2147/IJN.S38013
Video abstract
Point your SmartPhone at the code above. If you have a QR code reader the video abstract will appear. Or use:
http://dvpr.es/UdGa27
mailto:biswajit55@yahoo.comwww.dovepress.comwww.dovepress.comwww.dovepress.comhttp://dx.doi.org/10.2147/IJN.S38013http://qrcode.kaywa.com/img.php?s=8&d=http%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DzCQHiMUIEnghttp://dvpr.es/UdGa27.qrcodehttp://dvpr.es/UdGa27
International Journal of Nanomedicine 2012:7
such as nanotubes,6 nanorods,7 and nanoparticles.8 Gold
nanoparticles provide promising scaffolds for drug and gene
delivery. Their unique features, such as tunable core size,
monodispersity, large surface-to-volume ratio, and easy
functionalization with virtually any molecule or biomole-
cule enable their effective targeting, transport, and tuning
of delivery processes.9 Gold nanoparticles are the preferred
delivery system because of their relatively lesser intrinsic
toxicity toward the normal cell.9
Nanoparticles consisting of gold offer enhanced absorp-
tion and scattering, good biocompatibility, facile synthesis,4
and conjugation to a variety of biomolecular ligands, anti-
bodies, and other targeting moieties,5 making them suitable
for use in biochemical sensing and detection,10–12 medical
diagnostics, and therapeutic applications.13,14
Acquired immunodeficiency syndrome (AIDS) is a
disease of the human immune system caused by the human
immunodeficiency virus (HIV).4 This condition progres-
sively reduces the effectiveness of the immune system and
leaves the individual susceptible to opportunistic infec-
tion and tumor. It is transmitted through direct contact
of a mucous membrane or the bloodstream with a bodily
fluid containing HIV, such as blood, semen, vaginal fluid,
pre-seminal fluid, and breast milk.5 There is currently no
vaccine or cure for HIV or AIDS. The only known methods
of prevention are based on avoiding exposure to the virus
and antiretroviral treatment once affected. The antiviral
therapy has unpleasant side effects, including peripheral
neuropathy, acute pancreatitis, abdominal pain, diarrhea,
malaise, nausea, and fatigue. AIDS patients are generally
treated with nucleoside or nucleotide reverse transcriptase
inhibitors that inhibit reverse transcription by blocking the
reverse transcriptase enzyme responsible for conversion
from single-stranded RNA to double-stranded DNA in HIV.
Zidovudine, didanosine, zalcitabine, stavudine, lamivudine,
and abacavir are nucleoside analogs and tenofovir and
adefovir are nucleotide analogs used as reverse transcriptase
inhibitors for HIV infection. These drugs have severe side
effects at higher dose.
In the present study we used stavudine, which has a
short half-life and poor bioavailability. Nanoparticles are
used as a drug carrier for delivery of a drug to overcome
the problems of short half-life, poor bioavailability, and
strong side effects. In this study we developed, evaluated,
and compared a poly(d,l-lactic-co-glycolic acid) (PLGA)-
based nanoparticulate drug delivery system, with or without
gold nanoparticles, containing stavudine. We also studied the
uptake of nanoparticles by macrophages in vitro, since HIV
accumulates in macrophages during the early phase (first 1
to 2 years) of infection.6
In the present study, PLGA was used, since it is an FDA-
approved biodegradable polymer capable of drug release
in a sustained manner. In addition to the drug, gold nano-
particles were incorporated into the polymeric nanoparticle
matrices with the following expectations: first, since a gold
nanoparticle has a higher surface-to-volume ratio and gold
can hold many different molecules attached physically on
its surface, we wanted to see whether gold can help enhance
stavudine-loading in the nanoparticle; second, gold nano-
particles and their conjugates have been reported to inhibit
HIV transinfection;15 third, the presence of gold in PLGA
nanoparticles can be used for imaging to localize the pres-
ence of nanoparticles in organs and tissues in vivo, which is
the future program in the study.
Materials and methodsMaterialsStavudine was obtained from Cipla Ltd (gift sample)
(Baddi, India). PLGA (85:15; molecular weight [MW]
50,000–75,000), fluorescein isothiocyanate (FITC), chloro-
auric acid (HAuCl4), and trisodium citrate were purchased
from Sigma-Aldrich Corporation (Bangalore, India).
Polyvinyl alcohol ([PVA] MW 30,000–70,000) was obtained
from SD Fine Chem Ltd (Mumbai, India). Dichloromethane
(DCM) was purchased from E Merck (India) Ltd (Mumbai,
India). Disodium hydrogen orthophosphate and potassium
hydrogen phosphate were obtained from Process Chemical
Industries (Kolkata, India). All other chemicals used were
of analytic grade.
Preparation of polymeric drug-loaded nanoparticlesPVA solutions were prepared in 1.5% (1.5 g PVA in
100 mL water) and 2.5% (0.25 g of PVA in 10 mL water)
concentrations. We then dissolved 250 mg PLGA in 2 mL
DCM. The total amount of stavudine (Table 1) was dissolved
in 0.5 mL of 2.5% PVA. The drug solution (0.5 mL) was
then added drop-wise into 2 mL of the PLGA solution while
homogenizing, and the mixture was homogenized (WiseTis
homogenizer; Daihan Scientific Co Ltd, Seoul, South Korea)
at different speeds for different formulations (Table 1) for
4 minutes. This resulted in a water-in-oil (w/o) type of
emulsion. The emulsion was gradually added to 75 mL of
1.5% PVA solution and homogenized at the same speed
according to the formulation for 6 minutes, which produced a
water-in-oil-in-water (w/o/w) type of emulsion. The emulsion
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6050
Basu et al
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
was stirred at 130 rpm in a rotary vacuum evaporator (PBU-6;
Superfit, Mumbai, India) with a temperature-controlled water
bath (40°C) for 30 minutes for quick removal of the organic solvent (DCM). The emulsion was kept on a magnetic stirrer
overnight for complete removal of the DCM. The next day,
any particles that had formed were collected by centrifugation
at 15,000 rpm for 1 hour. To remove free drug, polymeric
particles were resuspended in water and centrifuged three
times at 15,000 rpm for 30 minutes each. Particles were
then freeze-dried (by prefreezing at −20°C overnight and lyophilizing at −40°C for 12 hours) in a lyophilizer (labora-tory lyophilizer; IIC Industrial Corporation, Kolkata, India)
and stored at 4°C. Formulations S1, S2, and S5 were prepared as described above, except that S3 and S4 were centrifuged
at 16,000 rpm and no drug was added to S5.
Preparation of gold nanoparticlesGold nanoparticles were prepared by citrate reduction of
HAuCl4 following the methods of Storhoff et al.7 All glass-
wares were first cleaned in aqua regia (three parts HCl, one
part HNO3), rinsed with nanopure water, and dried in a hot
air oven (Orion Industries, Kalka, India). An aqueous solu-
tion of HAuCl4 (1 mM, 500 µL) was brought to boiling while
stirring continuously with a glass rod, and the entire 50 mL
aliquot of 38.8 mM trisodium citrate solution was quickly
added, which resulted in a color change of the solution from
pale yellow to deep red. After the color change, the solution
was allowed to cool and was subjected to high-speed cen-
trifugation (3K30 Sigma Lab Centrifuge; Merrington Hall
Farm, Shrewsbury, UK) at 12,800 rpm at 4°C for 20 minutes. The gold nanoparticle pellet was then resuspended in water
at pH 7.0 after discarding the supernatant. The process was
repeated three times to eliminate the free citrate.8
Preparation of gold and drug-loaded polymeric nanoparticlesPVA solutions were prepared in 1.5% (by dissolving 1.5 g
PVA in 100 mL water) and 2.5% (by dissolving 0.25 g of PVA
in 10 mL water) concentrations. Colloidal gold solution
(1 mL) was added to the 2.5% PVA solution. PLGA
(250 mg) was dissolved in 2 mL DCM. The total amount
of stavudine (Table 1) was dissolved in 0.5 mL of the 2.5%
PVA–gold mixture. This drug solution (0.5 mL) was added
drop-wise into 2 mL of the PLGA solution, and the mixture
was homogenized at different speeds for different formula-
tions (Table 1) for 4 minutes. The preparation method was
then continued as described previously. To remove uncoated
gold nanoparticles from suspension after encapsulation, the
mixture was centrifuged at 6000 rpm for 1 hour. The super-
natant was decanted carefully, and coated nanoparticles were
separated from the supernatant by centrifugation at 15,000
rpm for 1 hour and then washed three times by centrifuging
at 15,000 rpm and resuspending in water. Formulations S2
and S4 were prepared as described above, except that S4 was
centrifuged at 16,000 rpm.
Nanoparticles with FITCStavudine-loaded nanoparticles were prepared with a fluo-
rescent probe (FITC) to study the uptake of nanoparticles
by specific cells. FITC-loaded nanoparticles were prepared
essentially as described above, except that an FITC stock
solution (0.4% weight [w]/volume [v] FITC in absolute
ethanol) of 100 µL was added to the polymeric phase dur-ing the preparation of the primary emulsion. The ratio of the
fluorophore to nanoparticles taken was 1:80. Preparation was
then continued as described above.
Evaluation and characterization of stavudine-loaded nanoparticlesDrug–excipient interactions by Fourier transform infrared spectroscopy (FTIR)Stavudine, PLGA, PVA, gold nanoparticles, PLGA–PVA
combination, stavudine–PLGA–PVA combination, and
stavudine–PLGA–PVA–gold nanoparticles combination
were mixed with infrared-grade potassium bromide (KBr)
Table 1 Composition of experimental nanoparticles with their drug loading and entrapment efficiency
Formulation code
Stavudine:PLGA (mg:mg)
Presence of colloidal gold
Speed of homogenization (rpm)
Amount recovereda (mg)
Drug loadingb (%)
Entrapment efficiencyc (%)
S1 20:250 No 15,000 165.4 5.69 76.83S2 20:250 Yes 15,000 187.2 5.08 68.57S3 25:250 No 16,000 150 7.93 87.23S4 25:250 Yes 16,000 186.6 6.15 67.63S5 0:250 No 15,000 152 NA NA
Notes: aRecovery (mg) = amount of lyophilized formulation obtained (mg) out of total amount of drug and excipients used (mg); bDrug loading (actual) (%) = Amount of drug in nanoparticles × 100/Amount of nanoparticles obtained; cDrug entrapment efficiency (%) = Drug loading (actual) (%) × 100/Drug loading (theoretical) (%).Abbreviations: NA, not applicable; PLgA, poly(d,l-lactic-co-glycolic acid).
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6051
gold–stavudine–PLgA nanoparticles
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
and compressed into pellets by applying 5.5 t of pressure in a
hydraulic press. The pellets were scanned over a wavenum-
ber range of 4000 cm–1 to 400 cm–1 in an FTIR spectrometer
(MAGNA-IR 750; Nicolet Instruments Corporation, Madison,
WI).
Morphology of nanoparticles by field-emission scanning electron microscopy (FESEM)The external morphology of nanoparticles of different
formulations was analyzed by FESEM. The freeze-dried
particles were spread onto metal stubs and platinum coat-
ing applied by using an ion-sputtering device. The coated
particles were then examined under FESEM (JSM 6100;
JEOL, Tokyo, Japan).
Determination of presence of gold in polymeric nanoparticle by energy dispersive X-ray spectroscopy (EDX)EDX was part of the FESEM system. SEM-EDX, which uses
an SEM system, helps determine the chemical composition
of a specimen, and we used this to determine the presence of
gold in stavudine–gold-loaded polymeric nanoparticles.
Determination of size distribution, polydispersity index (PDI), and zeta potentialSize distribution, PDI, and zeta potential were measured by a
Zetasizer Nano ZS (0.6 nm to 6000 nm) with DTS software
version 4.0 (Malvern Instruments Ltd, Malvern, UK). The
freeze-dried formulations were suspended in double-distilled
water and then poured into a glass cuvette and analyzed. For
particle size measurement, dynamic light scattering is used,
and the software collects and interprets data on particle size
and zeta potential and calculates the average size and PDI by
using the intensity, volume, and number distribution. Mean
particle diameter was calculated from the measured size
distributions, and the PDI was calculated based on the size
range present in the suspension as determined in a Zetasizer
Nano ZS (Malvern Instruments Ltd).
Drug content and entrapment efficiency studyExactly 2 mg of each product sample was placed in separate
2 mL Eppendorf microcentrifuge tubes. A prepared solution
of 5% w/v sodium dodecyl sulfate in 1 mL 0.1 M NaOH solu-
tion was added to each tube with a micropipette. The tubes
were placed in an incubator shaker (Somax Incubator Shaker;
Shenzhen Pango Electronic Co, Ltd, Shenzhen, China) at 120
rpm for 3 hours at 37°C. The tubes were then centrifuged for
10 minutes at 5000 rpm and the supernatant liquid collected
with a micropipette. The absorbance of drug in solution was
read with an ultraviolet absorption spectroscope (Beckman
Instruments, Fullerton, CA) at 266 nm against a blank con-
taining 5% w/v sodium dodecyl sulfate in 0.1 M NaOH.
Drug concentrations were determined from the calibration
curve. The drug-loading and drug entrapment efficiencies
were calculated using the following formulae:
Drug loading (theoretical) (%)
= Amount of drug taken to prrepare nanoparticles 100
Amount of PLGA + drug taken
×
(1)
Drug loading (actual) (%)
= Amount of drug in nanoparticless 100
Amount of nanoparticles obtained
×
(2)
Drug entrapment efficiency (%)
= Drug loading (actual) (%) 100
Drug loading (theoretical) (%)
×.
(3)
Drug release studyTo determine drug release at the different time points, 5 mg
stavudine-loaded nanoparticles of different formulations
were suspended in 1 mL phosphate-buffered saline ([PBS]
pH 7.4) in prelabeled microcentrifuge tubes and kept in an
incubator shaker (Somax Incubator Shaker) at 37°C with constant shaking at 72 rpm after brief vortexing. Each for-
mulation was processed in triplicate, and samples were kept
for specific periods of time up to 63 days. At any particular
time point, only the sample for analysis was removed from the
shaker, centrifuged at 15,000 rpm for 30 min at 4°C, and drug from the supernatant was analyzed at a wavelength of 266 nm
with a UV-Vis spectrophotometer (Beckman Instruments).
The percentage of drug release was calculated as:
Drug release (%)
= Amount of drug released
Amount of drug looaded in 5 mg of nanoparticle100×
(4)
Drug release kinetics studyTo understand the release kinetics, data obtained from in
vitro drug release studies were plotted in various kinetic
models: zero order as cumulative amount of drug released
versus time; first order as logarithmic value of cumulative
percentage of drug remained versus time; Higuchi model as
cumulative percentage of drug released versus square root
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6052
Basu et al
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
of time; Korsmeyer–Peppas model as logarithmic value of
cumulative percentage of drug released versus logarithmic
value of time; and Hixson–Crowell model as cube root per-
centage drug remained versus time.16 The in vitro drug release
data were also fitted to the Hopfenberg model.17,18
In vitro cellular uptake studyMacrophages at a density of 2.5 × 105 cells/mL were sus-pended in serum-free Roswell Park Memorial Institute
1640 medium (Sigma-Aldrich Corporation).15 A cell suspen-
sion of 200 µL was placed in eight-well tissue culture plates, each well of which contained 800 µL medium. Cells were allowed to adhere for 2 hours in a CO
2 incubator with a sup-
ply of 5% CO2 at 37°C. The medium was then removed, and
the wells were washed twice with serum-free medium. The
adherent macrophages were incubated with FITC–stavudine–
nanoparticles (S4) at different concentrations (300 ng/mL
and 500 ng/mL of medium) in 500 µL of fresh medium. After 4 hours, wells containing macrophages were washed
twice with serum-free medium, observed under confocal
laser scanning microscopy (LSM MAT 510; Carl Zeiss, Jena,
Germany), and photographs taken.
Intensity of the fluorescence of the individual mac-
rophages was quantified with a defined line and color
region, analyzed using Zen software (Zen Software Ltd,
Manchester, UK), and fluorescence intensity histograms
generated (not shown). Image software was used to correct
threshold and background levels19 and to quantify the degree
of fluorescence20 and the quantity of nanoparticles in cells.
The quantity of gold nanoparticles within the cells was
determined using the atomic emission spectroscopy of acid-
digested cells.21 The method ensures a highly sensitive analysis,
and a comparison of gold nanoparticle uptake by all exposed
cells has been successfully used to quantify the uptake of the
unmodified gold nanoparticle in the range of parts per billion.
At 4 hours incubation time, cell aliquots were washed twice
with cold PBS (pH 7.4) by centrifugation at 4000 rpm for
10 minutes at 4°C and resuspended in cold PBS. The pellet was then collected and analyzed using the method described.21
ResultsDrug–excipient interactions provide information on the stabil-
ity of the drug in formulation, the drug-release pattern from
the formulation, and the lag time of drug release.10 Among
the various methods available, FTIR spectroscopy provides
a methodology for examining interactions between various
functional groups present in drug and excipients. The FTIR
spectrum of the pure drug and individual FTIR spectrum of
PLGA, PVA, PVA–PLGA, and PVA–PLGA–drug are shown
in Figure 1. Comparing the results in Figure 1A with those
of Figure 1B–E, it can be seen that there was minor shift-
ing of some of the peaks between wavenumbers 3600 cm−1
and 2800 cm−1, 1700 cm−1 and 1800 cm−1, and 750 cm–1 and
500 cm−1. Wavenumber 3600 cm−1 to 3800 cm−1 is the infra-
red absorption frequency stretching vibration zone of strong
intensity CH3, CH
2, CH, medium intensity = CH and = CH
2,
strong intensity OH (hydrogen bonded), variable intensity OH
(free), medium intensity CH (aldehyde), and weak intensity
NH (1º amine) and NH (2º amine).22 Wave number 1700 cm−1
to 1800 cm−1 is the strong intensity stretching vibration zone
of C=O (saturated aldehyde), C=O (saturated ketone), cyclo-pentanone, and cyclobutanone.22 Wave number 750 cm−1 to
500 cm−1 is the bending vibration zone of weak intensity CH2
rocking, medium intensity out of plane = CH2, medium inten-
sity cis -RCH=CHR, strong intensity CH deformation, strong to medium intensity CH bending and ring puckering, weak
OH out of plane bending, and variable NH2 and NH wagging.
In stavudine, CH3, C=O, NH, N, OH are the reactive
groups, whereas in PLGA, CH3, C=O, and =O reactive
groups are present. For PVA, OH and H are the predomi-
nant reactive groups. Thus, there might be some physical
interactions between the functional groups of the drug and
the excipients that resulted in a minor shifting of the peaks.
Formation of a weak hydrogen bond, interaction due to van
der Waals force, or dipole–dipole interaction among these
functional groups might be responsible for these physical
interactions. The presence of the characteristic peaks of the
drug and the excipients suggests that there was no chemical
interaction. Further, FTIR spectra of the drug with gold mix-
ture and of the drug with gold and other excipients showed
no shifting of the characteristic peaks (data not shown). Thus,
FTIR spectroscopic data suggest that there was no chemical
interaction between the drug and the excipients, indicating
that the polymer is suitable for formulation of the anti-AIDS
drug. However, a few physical interactions occurred among
some functional groups of the drug and the excipients. These
might have been responsible for sustained release of the drug
from the formulations and might provide information on the
spherical structure of the nanoparticles.
Particles were mostly in the nanoscale range with a smooth
surface; however, there were some particles larger than nano-
size (Figure 2). The Z-average of S1–S5 formulations were
1365 nm, 1136 nm, 211 nm, 201 nm, and 2038 nm, respectively
(Table 2); polydispersity indices of the S1–S5 formulations
were 0.102, 0.109, 0.424, 0.376, and 0.046, respectively.
Particle size distribution (by percentage intensity) of the
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6053
gold–stavudine–PLgA nanoparticles
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
experimental formulations are shown in Figure 3. Zeta poten-
tials of S1–S5 were −12.6, −8.16, −27.8, −19.6, and −17 mV, respectively (Table 2). The dispersant refractive index, vis-
cosity, and dispersant (water) dielectric constant were 1.33,
0.8872, and 78.5, respectively, at 25°C. The incorporation of gold in nanoparticles was assessed from the EDX data, which
showed the presence of gold particles in gold-encapsulated
polymeric nanoparticles (Figure 4) as compared to gold nano-
particles (Figure 5) alone.
Maximum conductivity was recorded for S2 (0.034),
followed by S1 (0.011) and S5 (0.012) (Table 2). However,
conductivities were only slightly lower for S3 and S4.
04000 [comment]sample name S 3000
50
100110
2000
Wavenumber [cm−1]
Wavenumber [cm−1]
Wavenumber [cm−1]
Wavenumber [cm−1]
Wavenumber [cm−1]
E Stavudine, PLGA and PVA
D Mixture of PLGA and PVA
C PVA
B PLGA
A Stavudine
1000 400
10
20
4000 PLGA
%T
%T
%T
%T
3000
80
60
40
100
2000 1000 400
400076
80
3000
90
100
2000 1000 400
400040
3000
80
60
100
2000 1000 400
400077
80
3000
90
100
2000 1000 400
1
2 3
4
6
7
8
910 11
12
1314
1516
17
185
16
1514
13
1211
109
865
43
21
7
1 2
3 45 6
7
98
765432
1
1
2
34 5
6 7
8 9
Figure 1 FTIR spectra of (A) stavudine; (B) PLgA; (C) PVA; (D) PVA and PLgA mixture; and (E) stavudine, PVA, and PLgA mixture.Abbreviations: FTIR, Fourier transform infrared spectroscopy; PLgA, poly(d,l-lactic-co-glycolic acid); PVA, polyvinyl alcohol.
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6054
Basu et al
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
The drug-loading values were expressed in terms of the
quantity of drug entrapped in the formulations,11 and the drug
entrapment efficiency was associated with the percentage of drug
entrapped with respect to theoretical drug loading in a particular
formulation. S3 had the highest drug content and drug entrap-
ment efficiency among the formulations S1 to S4 (Table 1).
The in vitro drug release profile of stavudine from the
PLGA-based particulate drug formulations is shown in
Figure 6. The cumulative amount of drug release varied
between 65% and 72% of the actual drug content in the for-
mulations for 63 days. Gold along with stavudine-loaded poly-
meric nanoparticles (S4) prepared at a higher homogenization
10 µm 2 µmEHT = 20.00 kV WD = 17.0 mm Signal A = SE1 CGLEHT = 20.00 kV WD = 17.0 mm Signal A = SE1 CGL
IACS
10 µm1 µm
SEI 5.0 kV x30.000 100 nm WD 8.0 mm IACS SEI 5.0 kV x100.000 100 nm WD 8.0 mm
A B
C D
E F
Figure 2 FESEM photographs of formulations. (A) Formulation S1 prepared at 15,000 rpm shows that the particles were of micrometer sizes. (B) Formulation S2 prepared at 15,000 rpm shows that the particles were in micron range. (C) Formulation S3 prepared at 16,000 rpm shows that the particles were in nanometer range. (D) Formulation S4 prepared at 16,000 rpm shows that the particles were in nanometer range. (E) Formulation S5 prepared at 15,000 rpm shows that the particles were mostly in micrometer range. (F) SEM photograph of the gold nanoparticles shows that the particles were in submicron range with variable sizes.Abbreviations: FESEM, field-emission scanning electron microscopy; SEM, scanning electron microscopy.
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6055
gold–stavudine–PLgA nanoparticles
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
speed (16,000 rpm) showed a higher percentage of drug release
in 63 days compared to S3 during the same time period.
Data related to drug release as assessed by different
kinetic models16 showed more linearity in R2 values (Table 3)
in Higuchi, Korsmeyer–Peppas, and Hopfenberg plots. This
suggests anomalous diffusion of drug as well as erosion of
the matrix type formulations.
Based on the size and drug-loading in gold containing
PLGA nanoparticles, S4 was selected for the nanoparticle
uptake study. Nanoparticle uptake by macrophages was
observed in vitro after treatment of nanoparticles at two dif-
ferent concentrations for 4 hours (Table 4; Figure 7). As the
nanoparticle concentration increased, nanoparticle uptake
increased. About 34% and 45% of the nanoparticles were
internalized in 4 hours at nanoparticle doses of 300 ng/mL
(gold content 450 pg/0.5 × 105 cells) and 500 ng/mL (gold content 867 pg/0.5 × 105 cells), respectively, as assessed by fluorescence intensity.
DiscussionStavudine-loaded PLGA nanoparticles and stavudine with
colloidal gold loaded in PLGA nanoparticles were presented
in this research work. PLGA nanoparticles were formulated
using an emulsification solvent evaporation technique. PVA
was selected as an effective stabilizer12 and was used to sta-
bilize the emulsion since it also helps form relatively small
and uniform-sized particles.13,14
FTIR spectroscopy was used for the drug–excipients
interaction study to identify interactions10 at the functional
group level. No chemical interaction was detected between
the drug and the excipients since there was no shifting of
individual characteristic peaks. However, a few physical
interactions were found among some functional groups of
the drug and the excipients, and these interactions might be
responsible for the spherical structure of the nanoparticles
and their sustained drug release.
The experimental particles have smooth surface, and
particles prepared at 15,000 rpm were larger compared
to particles prepared at 16,000 rpm. The data suggest that
increasing homogenization speed results in decreases to
average particle diameters. Particles prepared at higher
homogenizing speed were in the nanoscale range and were
Table 2 Size distribution (Z-average [nm]), polydispersity index, zeta potential, and conductivity of experimental nanoparticulate formulations
Formulation code
Z-average (nm)
PDI Zeta potential (mV)
Conductivity (mS/cm)
S1 1365 0.102 −12.6 0.0117S2 1136 0.109 −8.16 0.0346S3 211 0.424 −27.8 0.00894S4 201 0.376 −19.6 0.00882S5 2038 0.046 −17 0.0125
Abbreviation: PDI, polydispersity index.
1000010001001010.10
10
Inte
nsi
ty (
%)
Size (d·nm)
Size distribution by intensity
20
30
Record 51: S5
1000010001001010.10
1
2
3
4
Inte
nsi
ty (
%)
Size (d·nm)
Size distribution by intensity
5
6
7
Record 50: S4
1000010001001010.10
10In
ten
sity
(%
)
Size (d·nm)
Size distribution by intensity
20
30
Record 49: S3 1
1000010001001010.10
10
Inte
nsi
ty (
%)
Size (d·nm)
Size distribution by intensity
20
30
Record 48: S2 2
1000010001001010.10
10Inte
nsi
ty (
%)
Size (d·nm)
Size distribution by intensity
20
30
40
Record 47: S1 1
A
B
C
D
E
Figure 3 Size distribution of different formulations. (A) S1 (by intensity); (B) S2 (by intensity); (C) S3 (by intensity); (D) S4 (by intensity); (E) S5 (by intensity).
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6056
Basu et al
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
homogeneously distributed. Encapsulation of colloidal gold
reduced the particle size even further. The incorporation
of gold in the nanoparticles was confirmed by the EDX
data.
An earlier study showed that polydispersity variation is
present from 100 nm to as large as 20 µm.23 In our study, polydispersity indices of the formulations varied widely.
The maximum PDI value was obtained for S5. The PDI
value was greater for all formulations with gold particles,
suggesting that the value was enhanced due to the presence
of the cationic metal.
Particles had negative zeta potentials. The resulting nega-
tive charges were caused by the dissociation of the hydrogen
ion from the carboxyl group (–COOH) in the PLGA chain.
Addition of PVA to the formulation also conveyed a negative
zeta potential to the resulting nanoparticle, further influenc-
ing both particle stability and the cellular uptake ability of
the nanoparticles.24,25 The presence of gold particles lowered
the zeta potentials of the formulation since gold is cationic.26
Particles with zeta potentials greater than +30 mV and less than −30 mV are normally considered to form stable colloidal dispersion.10 If the magnitude of the zeta potential decreases,
particle aggregation may occur, and this would cause a rapid
precipitation of suspended nanoparticles. Our results indicate
that all the formulations were unstable in terms of rapid
precipitation in the colloidal state. The zeta potential values
suggest that S3 had greater stability compared to the others
in the dispersion state. S2 was the least stable in terms of
00 1 2 3 4
Full scale 470 cts cursor; –0.005 (976 cts)5
Energy (keV)
Co
un
ts
6 7 8 9 10 11
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
420
440
460
O
Ca
Na
Mg
Au
Au
Si
Au
Ca
Au
Au
Au Au
Ca
Figure 4 EDX data showing the presence of gold particles in gold-encapsulated polymeric nanoparticles.Abbreviation: EDX, energy dispersive X-ray spectroscopy.
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6057
gold–stavudine–PLgA nanoparticles
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
00 1 2 3 4
Ca
Au
Au
Na
O
C
Ca
SiAu
Ca
AuAu
Au Au
Full scale 470 cts cursor; –0.005 (976 cts)5Energy (keV)
Co
un
ts
6 7 8 9 10 11
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
420
440
460
Figure 5 EDX data of colloidal gold nanoparticles alone.Abbreviation: EDX, energy dispersive X-ray spectroscopy.
80
70
60
50
40
30
20
10
00 10 20
S1 S2 S3 S4
30
Release study of stavudine from PLGA-based nanoparticles inphosphate-buffered saline at pH 7.4
Time (days)
Cu
mu
lati
ve p
erce
nta
ge
rele
ase
40 50 60 70
Figure 6 Release profile of didanosine from different formulations in phosphate-buffered saline (pH 7.4).Note: Data show means (n = 3).
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6058
Basu et al
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
forming a stable suspension, and these formulations should
be stored in a lyophilized condition and reconstituted in an
aqueous vehicle immediately before use.
Gold nanoparticles have a strong affinity toward sulfur
of the sulfhydryl (–SH) group through covalent interaction.
However, the most intriguing factor is the interaction between
amine and gold nanoparticles19 as this type of interaction
makes the gold nanoparticles more flexible upon attach-
ment and release. Any electronic perturbation can tune the
attachment or release of the amine-containing molecule (ie,
the drug) from the gold nanoparticle surface.26 In the present
study drug molecules were released relatively faster from
the PLGA nanoparticles containing gold. There was no
physicochemical interaction detected between the drug and
the gold nanoparticles, and the presence of gold in the PLGA
nanoparticles reduced the size (Z-average) of the particles.
Thus, the presence of gold in PLGA nanoparticles might have
shortened the length of the drug diffusion pathways thereby
releasing the drug relatively faster.
The drug-loading and the drug entrapment efficiencies in
the formulated particles were enhanced by using an optimum
amount of drug in preparatory steps27 and by using the proper
solvent or solvent mixture.20 The drug entrapment was gradu-
ally increased with the decreasing size of nanoparticles. Our
results showed that drug content was higher in drug-loaded
polymeric nanoparticles without gold compared to nanoparti-
cles with colloidal gold. No physical interaction, as assessed by
FTIR, was found between drug and gold. Gold nanoparticles
allow display of a large number of carbohydrates and other
similar molecules with a high density and multifunctionality
by the simultaneous incorporation of different ligands in a
single gold cluster in a controlled manner.28 However, the
lack of any distinct physicochemical interaction between the
drug and the PLGA suggests that no such phenomenon took
place. The presence of nanoscale-size gold particles within
the polymeric nanoparticles containing drug results in less
space available for the remaining drug compared to the nano-
particle containing drug without gold. Thus, the drug content
was less with the polymeric nanoparticle containing gold. In
addition, the presence of gold enhances the total weight of the
nanoparticles, which in turn reduces the percentage of drug
loading compared to the formulations without gold since the
percentage of drug loading is 100 times the amount of drug
present per unit quantity of nanoparticle. Thus, in spite of
the presence of gold nanoparticles, which have an increased
surface area to volume ratio, polymeric nanoparticles con-
taining gold appeared to accommodate less drug molecules.
In addition, drug content in the nanoparticles increased with
the incorporation of more drug in the formulation. However,
due to time constraints we could not determine the optimum
amount of drug to achieve maximum drug loading.
In vitro drug release profiles of stavudine from PLGA
nanoparticles showed that the cumulative percentage of drug
release was about 70% of the drug content of the formula-
tions in 63 days. The results support a steady and slow drug
release from the experimental nanoparticles. In vitro release
Table 3 Data of drug release kinetics of various formulations
Kinetic model Formulation S1 Formulation S2 Formulation S3 Formulation S4
Zero order R2 = 0.890 K0(µg mL
−1 h−1) = 1.0257R2 = 0.917 K0(µg mL
−1 h−1) = 0.9542R2 = 0.886 K0(µg mL
−1 h−1) = 0.8198R2 = 0.855 K0(µg mL
−1 h−1) = 0.8341First order R2 = 0.966
K1(h−1) = −0.0081
R2 = 0.973 K1(h
−1) = −0.0072R2 = 0.931 K1(h
−1) = −0.0063R2 = 0.908 K1(h
−1) = −0.0072Higuchi R2 = 0.986
KH(h−1/2) = 9.2194
R2 = 0.984 KH(h
−1/2) = 8.9496R2 = 0.951 KH(h
−1/2) = 7.8591R2 = 0.945 KH(h
−1/2) = 8.4073Korsmeyer–Peppas R2 = 0.825
n = 0.785R2 = 0.963 n = 0.657
R2 = 0.919 n = 0.519
R2 = 0.901 n = 0.453
Hixson–Crowell R2 = 0.945 KHC(µg
1/3 t−1) = −0.0233R2 = 0.958 KHC(µg
1/3 t−1) = −0.0212R2 = 0.918 KHC(µg
1/3 t−1) = −0.0189R2 = 0.889 KHC(µg
1/3 t−1) = −0.021Hopfenberg R2 = 0.9851
K(µg mg−1 µm−1)h−1 = 0.0092R2 = 0.9679 K(µg mg−1 µm−1)h−1 = 0.0104
R2 = 0.9143 K(µg mg−1 µm−1)h−1 = 0.0559
R2 = 0.9199 K(µg mg−1 µm−1)h−1 = 0.1139
Table 4 Nanoparticle (S4) uptake by macrophages
Serial no
Concentration of nanoparticles in suspension (ng/mL medium)
Effective concentration of drug (ng/mL medium)
Incubation time (hours)
Degree of nanoparticles uptake by macrophages (number of + symbols indicates the increasing degree)
1 300 17.28 4 +2 500 28.80 4 ++
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6059
gold–stavudine–PLgA nanoparticles
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine 2012:7
data of stavudine from the nanoparticles were tested by the
Higuchian kinetic model, which describes the release of drug
from an insoluble matrix as dependent on the time based
on Fickian diffusion. The release constant was calculated
from the slope of the appropriate plots, and the regression
coefficient was determined (Table 3). In vitro release of
stavudine was best explained by Higuchi’s equation as the
plots showed the highest linearity followed by first-order
kinetics. The corresponding plot for the Korsmeyer–Peppas
equation indicated a good linearity, particularly for S3 and
S4. The release exponent “n” had values in the range of
0.45 and 0.89, indicating that drug release was controlled by
anomalous diffusion, ie, diffusion and erosion.16–18 To confirm
the erosion mechanism of drug from the nanoparticles, the
Hopfenberg kinetic model17 was fitted to correlate the drug
release from surface-eroding polymers as long as the surface
area remains constant during the degradation process. The
linearity range (0.914 to 0.985) was good, suggesting that
the mechanism of drug release is controlled simultaneously
by diffusion and erosion.16–18
Targeting of stavudine-loaded nanoparticles to mac-
rophages has been assumed to more effectively kill HIV
as the virus accumulates in macrophages.6 In addition, the
nanoparticles were internalized by the macrophages in vitro,
as assessed by FITC fluorescence intensity. Increasing the
nanoparticulate concentration increased the fluorescent
intensity, at least for a period of 4 hours.
ConclusionStavudine was released from the experimental formulations
in a sustained manner over a prolonged period of time,
and this would minimize the frequency of dosing interval.
A faster drug-release pattern was observed for nanoparticles
containing colloidal gold compared to those without gold.
The presence of colloidal gold decreased drug loading in
PLGA nanoparticles. Drug release patterns appeared to fol-
low anomalous diffusion, and macrophages, an important
target group of cells in early HIV infection, were found to
internalize nanoparticles (S4), suggesting that dose-related
toxicity would be less. Polymer-encapsulated colloidal gold
nanoparticle uptake by macrophages provides support for
the use of gold nanoparticles as diagnostic and therapeutic
agents, but further studies are necessary to confirm this.
AcknowledgmentsWe are indebted to the Department of Biotechnology,
Government of India (Grant no BCIL/NER-BPMC/2012/650)
and the Indian Council of Medical Research (Grant no
45/20/2011/NAN/BMS) for partially funding the research
work.
DisclosureThe authors report no conflicts of interest in this work.
References1. Kreuter J. Evaluation of nanoparticles as drug delivery systems. I.
Preparation method. Pharm Acta Helv. 1983;58(7):196–209.2. Wang X, Wang Y, Chen Z, Shin DM. Advances of cancer therapy by
nanotechnology. Cancer Res Tre. 2009;41(1):1–11.3. Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE.
Biodegradable polymeric nanoparticles as drug delivery devices. J Controlled Release. 2001;70(1–2):1–20.
4. Sepkowitz KA. AIDS – the first 20 years. N Engl J Med. 2001;344(23): 1764–1772.
1. Only macrophages2. Only fluorescent molecules3. Macrophages with fluorescent molecules
300 ng/mL 500 ng/mL
1. Only macrophages2. Only fluorescent molecules3. Macrophages with fluorescent molecules
1 2
3
1 2
3
A B
Figure 7 Confocal microscopic image of macrophages treated with (A) stavudine nanoparticles (300 ng/mL of medium) for 4 hours, and (B) stavudine nanoparticles (500 ng/mL of medium) for 4 hours.
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
6060
Basu et al
www.dovepress.comwww.dovepress.comwww.dovepress.com
International Journal of Nanomedicine
Publish your work in this journal
Submit your manuscript here: http://www.dovepress.com/international-journal-of-nanomedicine-journal
The International Journal of Nanomedicine is an international, peer-reviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field. This journal is indexed on PubMed Central, MedLine, CAS, SciSearch®, Current Contents®/Clinical Medicine,
Journal Citation Reports/Science Edition, EMBase, Scopus and the Elsevier Bibliographic databases. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.
International Journal of Nanomedicine 2012:7
5. Weiss RA. How does HIV cause AIDS? Science. 1993;260(5112): 1273–1279.
6. Deneka M, Pelchen–Matthews A, Byland R, Ruiz-Mateos E, Marsh M. In macrophages, HIV-1 assembles into an intracellular plasma mem-brane domain containing the tetraspanins CD81, CD9, and CD53. J Cell Biol. 2007;177(2):329–341.
7. Storhoff JJ, Elghanian R, Mucic RC, Mirkin CA, Letsinger RL. One pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes. J Am Chem Soc. 1998; 120(9):1959–1964.
8. Patra HK, Banerjee S, Chaudhuri U, Lahiri P, Dasgupta AK. Cell selective response to gold nanoparticles. Nanomedicine. 2007;3(2): 111–119.
9. Boisselier E, Astruc D. Gold nanoparticles in nanomedicine: prepara-tions, imaging, diagnostics, therapies and toxicity. Chem Soc Rev. 2009; 38(6):1759–1782.
10. Mukherjee B, Patra B, Layek B, Mukherjee A. Sustained release of acyclovir from nano-liposomes and nano-niosomes. Int J Nanomedicine. 2007;2(2):213–225.
11. Bilensoy E, Gürkaynak O, Lale Doğanand A, Atilla Hıncal A. Safety and efficacy of amphiphilic ß-cyclodextrin nanoparticles for paclitaxel delivery. Int J Pharm. 2008;347(2):163–170.
12. Fu XD, Gao YL, Ping QN, Ren T. Preparation and in vivo evaluation of huperzine A-loaded PLGA microspheres. Arch Pharm Res. 2005;28(9): 1092–1096.
13. Mandal TK, Bostanian LA, Graves RA, Chapman SR. Poly(D,L-lactide-co-glycolide) encapsulated poly(vinyl alcohol) hydrogel as a drug delivery system. Pharm Res. 2002;19(11): 1713–1719.
14. Wang N, Wu XS, Li JK. A heterogeneously structured composite based on poly (lactic-co-glycolic acid) microspheres and poly (vinyl alcohol) hydrogel nanoparticles for long-term protein drug delivery. Pharm Res. 1999;16(9):1430–1435.
15. Arnáiz B, Martínez–Ávila O, Falcon–Perez JM, Penadés S. Cellular uptake of gold nanoparticles bearing HIV gp120 oligomannosides. Bioconjug Chem. 2012;23(4):814–825.
16. Pattnaik G, Sinha B, Mukherjee B, et al. Submicron-size biodegradable polymer-based didanosine particles for treating HIV at early stage: an in vitro study. J Microencapsul. 2012;29(7):666–676.
17. Dash S, Murthy PN, Nath L, Chowdhury P. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol Pharm. 2010; 67(3):217–223.
18. López-Gasco P, Iglesias I, Benedí J, Lozano R, Teijón JM, Blanco MD. Paclitaxel-loaded polyester nanoparticles prepared by spray-drying technology: in vitro bioactivity evaluation. J Microencapsul. 2011; 28(5):417–429.
19. Shan M, Klasse PJ, Banerjee K, et al. HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells. PLoS Pathog. 2007; 3(11):e169.
20. Falcón-Pérez JM, Nazarian R, Sabatti C, Dell’Angelica EC. Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3. J Cell Sci. 2005;118(Pt 22):5243–5255.
21. Freese C, Gibson MI, Klok HA, Unger RE, Kirkpatrick CJ. Size- and coating-dependent uptake of polymer-coated gold nanoparticles in pri-mary human dermal microvascular endothelial cells. Biomacromolecules. 2012;13(5):1533–1543.
22. Silverstein RM, Webster FX. Spectrometric Identification of Organic Compounds. 6th ed. New York: John Wiley & Sons; 1998:71–143.
23. Panyam J, Dali MM, Sahoo SK, et al. Polymer degradation and in vitro release of a model protein from poly (D,L-lactide-co-glycolide) nano- and microparticles. J Control Release. 2003;92(1–2):173–187.
24. Patil S, Sandberg A, Heckert E, Self W, Seal S. Protein adsorption and cellular uptake of cerium oxide nanoparticles as a function of zeta potential. Biomaterials. 2007;28(31):4600–4607.
25. Song C, Labhasetwar V, Cui X, Underwood T, Levy RJ. Arterial uptake of biodegradable nanoparticles for intravascular local drug delivery: results with an acute dog model. J Control Release. 1998;54(2):201–211.
26. Mocanu A, Cernica I, Tomoaia G, Bobos L-D, Horovitz O, Tomoaia-Cotisel M. Self-assembly characteristics of gold nanoparticles in the presence of cysteine. Colloids Surf A Physicochem Eng Asp. 2009; 338(1–3):93–101.
27. Sehra S, Dhake AS. Formulation and evaluation of sustained release microspheres of poly-lactide-co-glycolide containing tamoxifen citrate. J Microencapsul. 2005;22(5):521–528.
28. Ojeda R, de Paz JL, Barrientos AG, Martin-Lomas M, Penadés S. Preparation of multifunctional glyconanoparticles as a platform for potential carbohydrate-based anticancer vaccines. Carbohydr Res. 2007;342(3–4):448–459.
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
Dovepress
6061
gold–stavudine–PLgA nanoparticles
http://www.dovepress.com/international-journal-of-nanomedicine-journalhttp://www.dovepress.com/testimonials.phphttp://www.dovepress.com/testimonials.phpwww.dovepress.comwww.dovepress.comwww.dovepress.comwww.dovepress.com
Publication Info 2: Nimber of times reviewed: